HIV-1 causes a systemic infection of the human immune system, using the CD4 membrane protein expressed by T cells, dendritic cells, and macrophages as its primary entry receptor. Based on such tropism, lymphatic tissues are the primary sites of viral amplification, and likely also important sites of latent infection. It is currently not known how virus is transmitted between infected and uninfected cells in vivo. In vitro studies suggest that direct interactions between T cells, but also interactions between T cells and infected or noninfected dendritic cells and macrophages that trans-present HIV, greatly increase the efficiency of viral dissemination over transfer via virion release into the extracellular space. Here we propose to use multiphoton intravital microscopy (MP-IVM) for the temporospatially resolved visualization of the behavior of HIV-infected T cells in lymph nodes of humanized BLT mice. Employing a palette of recombinant HIV strains that confer fluorescence to infected cells we aim to obtain insight into the cellular mechanisms by which HIV-1 spreads among susceptible immune cells through direct cell-cell contacts in vivo. Aim 1 will explore the migratory characteristics of HIV-infected T cells in lymph nodes (subaim 1.1), and specifically the role of the viral proteins Nef and Env in any alterations of their motile behavior that are observed (subaims 1.2 and 1.3). We will also investigate if the infected T cells serve as cellular vehicles for HIV during the physiological process of T cell recirculation through secondary lymphoid organs, and thus contribute to its systemic dissemination (subaim 1.4). Aim 2 will examine the interactions of susceptible T cells with HIV+ cells in lymph nodes. Specifically we will measure the efficiency at which susceptible T cells are infected in HIV-infected lymph nodes (subaim 2.1) and analyze the frequency, duration, dynamics, and stoichiometry of T cell encounters with HIV-infected T cells (subaim 2.2) and with HIV+ dendritic cells (subaim 2.3). We will also explore whether and under what circumstances these interactions facilitate the intercellular transfer of cellular material including virus.